Honors Project May 2, 2013 By: Alyssa Rogers Mentor: Dr. Christopher Lane BROWN ALGAL DIVERSITY IN BERMUDA REVEALED USING MOLECULAR TOOLS

 Main objective is to integrate molecular and morphological data to produce thorough classifications of all of the marine macroalgae (red, green, and brown algae) of Bermuda A Brief Introduction to the Bermuda Seaweed Project Related species “look” identical, but aren’t Without comparative DNA sequences, impossible to distinguish with any certainty Crypsis: Convergent Morphologies

 A ‘fingerprint’ for identification of life  DNA barcodes are  Short  Universal  Variable enough to distinguish between species What is a DNA Barcode?

Why Bermuda? 32˚ 14-25’N 64˚ ’W 1000 km east of NC in the N. Atlantic 1350 km northeast of Bahamas Water temp range = °C Bermuda

 To figure out what is out there  Can be used as a reference and/or comparison for later studies  To identify species that may have been introduced to the area  To identify novel species that have not been seen before Why are Biodiversity Studies Important?

 Conduct a phylogenetic investigation to assess the diversity of the brown algae of Bermuda  How? Project Goals

 Evolutionarily distinct from the red and green algae who share an ancient common ancestor  Challenges when studying them due to their divergent DNA sequences and cellular chemistry compared to the reds and greens Brown Algae (Phaeophyceae)

Stypopodium zonale Canistrocarpus cervicornis Dictyopteris plagiogramma Lobophora variegata

 cox1 – cytochrome c oxidase subunit 1  rbcL – large subunit of ribulose 1, 5 bisphosphate carboxylase/oxygen ase (RUBISCO) Genes of Interest

 URI graduate student, Thea Popolizio, performed a year-long collection of samples in Bermuda  Variety of habitats (different locations, depths, etc.)  Various seasonal climates  Over 1500 samples of macroalgae were collected  About 20% of those samples are brown algae Collection of the Samples

Grinding of Samples Genomic DNA Extraction PCR Gel Electrophoresis Purification of PCR Sequencing Geneious v5.6.5 Analysis Methods and Materials

Using Geneious (V 5.6.5): Assembling Sequences

Using Geneious (V 5.6.5): Alignment of Consensus Sequences

Using Geneious (V 5.6.5): Creating Phylogenetic Trees

cox1 Data Analysis L. variegata

cox1 Data Analysis P. gymnospora

cox1 Data Analysis D. humifusa

A Closer Look at Dictyota

These analyses indicate that there are more species in the genera Lobophora, Padina, and Dictyota than currently documented for the Bermudian brown algae What Does All This Mean?

 Troubleshooting primers for the rbcL gene  The rbcL data currently being assembled agrees with the cox1 data  However, as of now, there is an insufficient amount of rbcL data to create a tree Problems Encountered rbcLrbcS Spacer

 cox1 and rbcL barcode data will be produced for the remaining brown algae samples  Barcode data will be produced for the red (cox1 and rbcL) and green (cox1 and tufA) Bermudian algae  Morphological data and molecular data will be combined to form robust classifications of the entire macroalgal flora of Bermuda Future Directions

 Dr. Christopher Lane  Thea Popolizio  Eric Salomaki  Tom Shamp  Funding:  National Science Foundation award # to the Lane Lab  The Rhode Island Genomics and Sequencing Center which is supported in part by the National Science Foundation (MRI Grant No. DBI and EPSCoR Grant Nos & EPS ), the US Department of Agriculture (Grant Nos and ), and the University of Rhode Island  Picture Credits:  algaebase.org Acknowledgements